Design, syntheses and evaluation of benzoylthioureas as urease inhibitors of agricultural interest

Article abstract of DOI:10.1039/c5ra07886e

Urea is one of the most used nitrogen fertilizers worldwide. However, occurrence of urea hydrolysis to ammonia and carbon dioxide on soil surface, catalyzed by soil ureases, considerably reduces nitrogen availability to crops. In this study, we describe the design, synthesis and screening of sixty five benzoylthioureas (BTUs) for their ability to inhibit purified jack bean and soil ureases. BTUs were readily obtained in one pot, two steps synthesis with no need of cumbersome procedures for product purification. In vitro assays revealed BTUs 11, 12, 14, 19-22 and 37 as the most active jack bean urease inhibitors. Such BTUs were found to be able to bind to both catalytic and allosteric sites of urease, acting therefore as mixed-type inhibitors. Out of 28 compounds that effectively inhibited soil ureases activity, BTUs 3, 6, 10, 12, 16, 19 and 22 were determined to be more potent than the reference inhibitor N-(butyl) thiophosphoric triamide (NBPT; 40%). The other 22 BTUs were as potent as NBPT on soil ureases. The temperature-tolerance of BTUs, along with their ability to inhibit soil ureases, makes of this class of compounds potential additive for urea-based fertilizers.

Full text of DOI:10.1039/c5ra07886e

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ARTICLE
Design, syntheses and evaluation of benzoylthioureas
as urease inhibitors of agricultural interest
Cite this: DOI: 10.1039/x0xx00000x
Tiago O. Britoa,†, Aline X. de Souzab,†, Yane C. C. Motab, Vinicius S. S. Moraisb,
Leandro T. de Souzab,c, Ângelo de Fátimad, Fernando Macedo Jra,*, Luzia V. Modolob,*
Received 00th January 2012,
Accepted 00th January 2012
Urea is some of the most used nitrogen fertilizers worldwide. However, occurrence of urea
hydrolysis to ammonia and carbon dioxide on soil surface, catalysed by soil ureases,
considerably reduces nitrogen availability to crops. In this study, we describe the design,
synthesis and screening of sixty five benzoylthioureas (BTUs) for their ability to inhibit
purified jack bean and soil ureases. BTUs were readily obtained in one pot, two steps
synthesis with no need of cumbersome procedures for product purification. In vitro assays
DOI: 10.1039/x0xx00000x
www.rsc.org/
revealed BTUs 11, 12, 14, 19ꢀ22 and 37 as the most active jack bean urease inhibitors. Such
BTUs were found to be able to bind to both catalytic and allosteric sites of urease, acting
therefore as mixedꢀtype inhibitors. Out of 28 compounds that effectively inhibited soil
ureases activity, BTUs
the reference inhibitor
3
,
6
,
10
, 12, 16, 19 and 22 were determined to be more potent than
N
ꢀ(butyl)thiophosphoric triamide (NBPT; 40%). The other 22 BTUs
were as potent as NBPT on soil ureases. The temperatureꢀtolerance of BTUs, along with
their ability to inhibit soil ureases, makes of this class of compounds potential additives for
ureaꢀbased fertilizers.
Introduction
esters of phosphoric acid, quinones and (poly)phenolic compounds
have been recognized as urease inhibitors.10,12,13 However, the best
Urea is some of the nitrogen (N) fertilizers most widely used in
agriculture because of its high N content (460 g kgꢀ1; Nꢀurea).
However, N loss by ammonia (NH3) volatilization is a primary
concern when urea is used in covering fertilization due to the activity
of soil ureases on this fertilizer.1 The average N losses via NH3
volatilization in Europe was found to be 14% of total urea applied to
soil2 with these losses being higher in tropical climate areas.3 Indeed,
several field studies in Brazil reported average N losses ranging from
20% to 60%.4ꢀ6 The N losses from NH3 volatilization are also an
environmental concern since NH3 can directly or indirectly
contribute to the increase of greenhouse gases in atmosphere and
contamination of streams and lakes via eutrophication.7 Several
substances, both natural and synthetic ones, have been evaluated for
their potential to inhibit urease as a strategy to increase urea
efficiency in agriculture and mitigate environmental pollution.7ꢀ10
Taking covering fertilization as an example, the partial inhibition of
soil ureases allows for slowing down urea hydrolysis on soil surface
increasing the chances of urea incorporation to the soil after rain or
irrigation episodes.11
results in soil have been achieved using urea analogs, particularly the
Nꢀ(butyl) thiophosphoric triamide (NBPT), which was found to bind
to active site of ureases after its conversion to the corresponding
oxoꢀderivative by soil microbiota.14,15 However, NBPT tends to lose
efficiency upon the time of storage and water exposure.16,17 The
NBPT efficiency is also reduced in acidic soils and under high
temperatures,17 conditions in which ureases have higher catalytic
activity.5 Therefore, the development of novel urease inhibitors of
agricultural interest is mandatory to overcome the drawbacks
presented by urease inhibitors currently on the market.
Thiourea derivatives are notable for a plethora of biological
activities such as antimicrobial18,19, antiꢀmalarial20, anticancer21,
antihypertensive22 and as inhibitor of the HIV reverse transcriptase.23
Moreover, the potential of N2ꢀsubstituted benzoylthioureas as urease
inhibitors of clinical interest was reported recently.24,25 Besides the
pharmacological properties, thiourea and derivatives have being
shown to possess herbicide26,27 and insecticide28 activities.
Additionally, the ability of regulating plant growth exhibited by
thiourea derivatives appears to be selective since such class of
compounds is generally well tolerated by crops.29,30
Heavy metal ions (Hg2+, Cd2+, Ag+ etc), boron compounds (boric
acid derivatives), organic molecules such as thiols, thiourea,
hydroxyurea, acylhydroxamic acids, amides, benzothiazoles and
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Considering the role of thiourea as urease inhibitor and the cmꢀ1): 3299 (amide NꢀH), 3025 (thiourea NꢀH), 1661 (C=O),
biological activities of its derivatives, this work focused on the 1254 (C=S). 1H NMR (400 MHz, DMSOꢀd6, ppm): 4.01 (s, 3H,
design and synthesis of 65 benzoylthioureas (BTUs) with potential OCH3); 6.80 (d,
to inhibit the ureolytic activity of ureases. The inhibitory urease (d, = 8.5 Hz, 1H); 7.46 (d,
properties were investigated in assays performed with purified jack Hz, 1H); 7.93 (d, = 7.5 Hz, 2H); 9.62 (s, 1H, OH); 11.15 (s,
J
= 8.5 Hz, 2H); 7.16 (t,
J
= 7.5 Hz, 1H); 7.28
J
J
= 8.5 Hz, 2H); 7.66 (t,
J
= 7.5
J
bean and soil ureases. Kinetic experiments were carried used to 1H, CONH); 12.35 (s, 1H, CSNH). 13C NMR (DEPT 135
address the mechanism of urease inhibition triggered by the best phase) (100 MHz, DMSOꢀd6, p.p.m.): 56.7(+) (OCH3);
BTUs.
112.8(+); 115.1(+); 119.5; 121.3(+); 125.8(+); 129.2; 131.2(+);
135.1(+); 155.9; 157.5; 165.2 (C=O); 177.8 (C=S). The data for
previously reported BTUs to which spectral values have not
been provided in the literature so far (NR; Tables 1 and 2) are
included as supplementary information.
Experimental section
Analysis grade solvents were used without further treatment
unless otherwise stated. Acetone was dried over molecular
sieves 3Å for c.a. 6ꢀ18 h before use. Melting points were
determined on a Microquímica MQAPF 302 hot plate
apparatus. Infrared spectra (400ꢀ4000 cmꢀ1) were recorded as
KBr discs on a Shimadzu FTꢀIR Model 8300 instrument. NMR
spectra were obtained on a Bruker spectrometer Model Avance
III operating at 400 MHz for 1H and 100 MHz for 13C using a 5
mm broadband probe. NMR resonances were registered using
CDCl3 or DMSOꢀd6 as solvents and TMS as internal standard.
Chemical shifts (δ in ppm) were referenced to the residual
solvent signals (CHCl3 in CDCl3 at δ 7.24; DMSO in DMSOꢀd6
Thermogravimetric measurements
Thermogravimetric measurements were carried out in a
Shimadzu TGAꢀ50 Thermogravimetric Analyzer according to
da Silva et al. (2013),32 except that nitrogen gas flow (50 mL
minꢀ1) was used instead of air flow. The weight change curves
were recorded from 30 oC to 500 oC.
Urease inhibition assay
The screening for identifying potential urease inhibitors was
done using the indophenol method.33 Each benzoylthiourea
1
at δ 2.50). The splitting of proton resonances in the reported H
NMR spectra are defined as singlet (s), doublet (d), triplet (t),
quartet (q) and complex pattern (m). Coupling constants (
reported in Hz.
(BTU) at final concentration of 500
µM was incubated in a
J) are
medium reaction buffered with 20 mM phosphate (pH 7.4) and
supplemented with 1 mM EDTA, 10 mM urea and 12.5 mU of
Canavalia ensiformis (jack bean) type III urease (Sigma).
Reactions were maintained at 25 °C for 10 to 15 min, followed
by addition of 0.5 volume of 1% w/v phenol in 5 ppm sodium
nitroprusside (SNP) and 0.7 volume of 0.5% w/v NaOH in
0.1% v/v NaOCl to interrupt enzyme activity. Reactions were
then incubated at 50 °C for 5 min prior the measurement of
absorbance at 630 nm to determine the amount of ammonium
Synthesis of benzoylthioureas (BTUs)
The N2ꢀsubstituted benzoylthioureas (BTUs)
1ꢀ65 were
synthesized according to methodology described elsewhere31,
with modifications. Briefly, suitable benzoyl chloride (11
mmol) was added to ammonium thiocyanate (440 mmol Lꢀ1) in
acetone (25 mL). The reaction mixture was heated under reflux
for 15 min and then cooled down to room temperature. An
acetone solution of suitable amine (11 mol Lꢀ1) was added to
this mixture, which was stirred under reflux for further 30 min
+
(NH4 ) formed. Hydroxyurea (HU) and thiourea (TU) were
used as references of urease inhibitors. Urease inhibition was
+
determined in terms of percentage of NH4 formed in BTUꢀ
(for BTUs
4ꢀ65) or under room temperature for 3 h (for BTUs
containing reactions in relation to total urease activity in
reactions devoid of inhibitor.
1ꢀ3
). The reaction mixture was then poured into crushed ice and
the resulting mixture was vigorously stirred. Each obtained
solid product was filtered off, washed with deionized water and
then recrystallized from ethanol or ethanol in acetone.
Kinetic assays with jack bean type III urease
The effect of BTUs synthesized on the kinetic parameters of
jack bean type III urease was investigated using a procedure
similar to that adopted for the in vitro screening, except that
Full data set for the novel BTUs are listed as follows.
N-(4-
Hydroxyphenylcarbamothioyl)-3-methoxybenzamide (BTU
59): Recrystallization from ethanol (74% yield). FTꢀIR (KBr,
cmꢀ1): 3358 (amide NꢀH), 3014 (thiourea NꢀH), 1663 (C=O),
1271 (C=S). 1H NMR (400 MHz, DMSOꢀd6, ppm): 3.85 (s, 3H,
different concentrations of BTUs (250 to 500 µM) and urea (1
to 32 mM) were employed in the reaction media. Each BTU
was used at concentrations necessary to inhibit the jack bean
urease ureolytic activity in the range from 30 to 40%. Kinetic
parameters of BTUꢀfree and BTUꢀcontaining reactions were
obtained using Hyper32 software. OriginPro 8 software was
used to obtain MichaelisꢀMenten hyperbolas and Lineweaverꢀ
Burk plots to assess the mechanism by which the BTUs inhibit
the ureolytic activity of urease.
OCH3); 6.80 (d,
J = 8.2 Hz, 2H); 7.21 (d, J = 8.2 Hz, 1H); 7.44
(m, 3H); 7.55 (m, 2H); 9.59 (s, 1H, OH); 11.49 (s, 1H, CONH);
12.42 (s, 1H, CSNH). 13C NMR (DEPTꢀ135 phase) (100 MHz,
DMSOꢀd6
119.4(+); 121.0(+); 126.0(+); 129.4; 129.6(+); 133.5; 155.8;
159.0; 168.0 (C=O); 179.0 (C=S). -(4-
,
ppm): 55.5(+) (OCH3); 113.2(+); 115.1(+);
N
Hydroxyphenylcarbamothioyl)-2-methoxybenzamide (BTU
64): Recrystallization from ethanol (82% yield). FTꢀIR (KBr,
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA07886E
Soil ureases activity assay
The effect of synthesized BTUs on the activity of soil ureases
was assessed using the salicylate method.34 Clayey dystrophic
Red Latosol (oxisol) soil was collected from Brazilian Cerrado
(19°28'01.2"S, 44°10'24.5"W). The physical features of the
collected soil were 6% coarse sand, 4% fine sand, 12% silt, and
78% clay and chemical analyses showed pH 6.3, 10 mg dmꢀ3
PMehlichꢀ1, 129 mg dmꢀ3 K, 4.4 cmolc dmꢀ3 Ca, 0.9 cmolc dmꢀ3
Mg, 0.1 cmolc dmꢀ3 Al, 2.6 cmolc dmꢀ3 H+Al, sum of bases of
5.6 cmolc dmꢀ3, 68% base saturation and organic matter of 2.5
dag kgꢀ1. Sieved soil (0.5 g; particles < 2 mm) were incubated
with 72 mM urea in the presence or absence of each BTU (500
The BTUs 2-65 (Tables 1 and 2) were prepared by reacting
primary amines with in situꢀproduced benzoyl isothiocyanate.31
Compounds
reflux while adducts
4
ꢀ
65 (Fig. 1) were obtained after 30 min under
and were formed after 3 h at room
2
3
temperature under stirring. The BTUs synthesized were
obtained in moderate to high yields (Tables 1 and 2). The BTU
1
was synthesized in a similar manner, except that NH4OH was
used instead of an amine. Measured melting points and/or
1
spectroscopic data (IR; H and 13C NMR) for the BTUs herein
synthesized were compared to those reported in the literature,
when available (Tables 1 and 2). The structures of the novel
BTUs 59 and 64 were confirmed by spectroscopic techniques.
Strong C=O stretching absorption and weak NꢀH stretching
were observed for all compounds in IR spectra between 1700ꢀ
1660 cmꢀ1 and 3419ꢀ3011 cmꢀ1, respectively. A complex set of
bands, characteristic of the thioamide moiety were also
observed in accordance to data reported elsewhere.35 The C=S
stretching was observed between 1291ꢀ1236 cmꢀ1.
µ
M) at 37 oC for 1 h
. The activity of soil ureases was
interrupted by the addition of 1 M KCl in 10 mM HCl (5 mL).
After 30 min incubation at 25 °C, a supernatant fraction was
collected and added to a mixture constituted of 3.4% sodium
salicylate, 2.5% sodium citrate, 2.5% sodium tartrate and 120
ppm SNP. Systems were incubated for further 15 min at 25 °C
and under darkness. Then, 0.1 volume of 3.0% NaOH in 1.0%
sodium hypochlorite was added to each system following
incubation under darkness for 1 h at 25 °C and stirring (600
rpm). Spectrophotometric measurements were carried out at
Table 1. Synthetic approach for obtaining benzoylthioureas
+
660 nm to estimate NH4 formed in the reaction media. The
Nꢀ
based on the use of benzoyl chloride as a start material.
(butyl) thiophosphoric triamide (NBPT) was used as a
reference of urease inhibitor active on soil ureases.
R1
O
HN
O
O
R1-NH2
Me2CO
NH4SCN
Me2CO
N
S
Cl
NCS
H
Statistical analysis
Data obtained from experiments carried out with soil were
analyzed by oneꢀway ANOVA followed by a Stepwise contrast
BTU
1
2
3
4
5
6
7
8
R1
H
CH3
CH2CH3
C4H9
C6H11
C6H5
C6H5CH2
C6H5(CH2)2
4ꢀOMeꢀC6H4
4ꢀOHꢀC6H4
4ꢀ(CH3)ꢀC6H4
4ꢀ(C4H9)ꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀClꢀC6H4
4ꢀBrꢀC6H4
3ꢀOMeꢀC6H4
3ꢀOHꢀC6H4
3ꢀNO2ꢀC6H4
3ꢀClꢀC6H4
3ꢀBrꢀC6H4
2ꢀOMeꢀC6H4
2ꢀNO2ꢀC6H4
2ꢀClꢀC6H4
2ꢀBrꢀC6H4
2ꢀpyridinyl
2ꢀpyrimidinyl
Yield (%)a
50
Ref.
M.p. ( °C)
170
147
125
124
64
43
44
NR
44
45
46
47
48
49
50
49
NR
51
46
50
49
52
51
49
53
51
51
46
54
41
55
test (
P
< 0.001) using the R software.
38
58
58
90
79
80
86
92
96
94
79
93
97
85
87
70
89
99
85
92
64
74
86
71
Results and discussion
146
121
105
145
160
156
114
179
136
144
105
184
158
125
129
150
146
142
134
137
176
Preparation of benzoylthioureas (BTUs)
The design of BTUs was based on thiourea (TU) structure, a
known urease inhibitor.12 Then, N1,N2ꢀdisubstituted thioureas
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
bearing cores
A and B constituted of aliphatic, aromatic,
heteroaromatic, benzylic and homobenzylic groups were
synthesized to check if the structural modifications improved
the ability of thiourea to inhibit ureases (Fig. 1).
X
H2N
NH2
X = O Urea (urease substrate)
X = S Thiourea (urease inhibitor)
A
H
CH3
CH2CH3
tert-Butyl
R = H, CH3, tert-butyl, MeO,
NO2, OH, Br or Cl
S
O
A
73
N
N
B
H
H
N
aValues correspond to the overall yield of twoꢀstep reactions. M.p.,
melting point as compared to that reported elsewhere (if available).
NR, not yet reported so far.
Benzoylthioureas
(Potential new inhibitors)
N
N
R = H, MeO, NO2 or Cl
B
1
All detected H and 13C NMR peaks were consistent with the
Fig. 1. Strategy used for the design of urease inhibitors of
agricultural interest based on benzoylthiourea moiety.
presence of the corresponding aliphatic or aromatic group in
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DOI: 10.1039/C5RA07886E
BTUs structure. The CONꢀ
H
and CSNꢀ
H
protons peaks were
(27ꢀ65; Table 2) in 40% to 99% yield. Interesting features of
observed as broad singlets between δH 8.69ꢀ12.35 and δH 9.85ꢀ the herein reported BTUs preparation include (i) the starting
13.60, respectively. The 13C NMR peak between δC 176.8ꢀ182.1 materials are commercially available, (ii) products are obtained
was assigned to C=S carbon present in each BTU structure. in oneꢀpot reaction carried out in only two steps with no need of
Furthermore, carbonyl signal was observed in δC 164.9ꢀ168.6 further purification of benzoyl isothiocyanates formed as
range, which is the typical region for C=O of intermediates, (iii) products are easily purified from
benzoylthioureas.31
recrystallization and (iv) products are solid, facilitating storage
and transportation. These BTU properties are particularly
interesting when compared to those of N-(butyl) thiophosphoric
triamide (NBPT), a urease proinhibitor commonly used as
additive in ureaꢀbased fertilizers. Differently from the BTUs,
NBPT is difficult to handle because it is obtained as a waxy
and sticky material.17
Table 2. Synthetic approach for obtaining benzoylthioureas
based on the use of substitutedꢀbenzoyl chlorides as start
materials.
R1
O
O
O
HN
R1-NH2
Me2CO
NH4SCN
Me2CO
Cl
NCS
N
S
H
Inhibitory effect of BTUs on jack bean urease activity
R2
BTU
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
R2
R2
R1
C6H5CH2
R2
Yield (%)a
49
76
68
95
83
73
73
77
81
89
89
82
60
76
73
43
77
99
77
99
99
68
72
79
75
78
98
94
81
95
82
76
94
96
78
69
95
82
76
M.p.
Ref.
56
57
NR
58
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
59
An in vitro screening was carried out with 65 BTUs in the
presence of purified jack bean type III urease and urea to
investigate the potential of these synthesized compounds to
inhibit the ureolytic activity of ureases. Hydroxyurea (HU) and
thiourea (TU), commonly used in in vitro screenings36ꢀ39, were
employed as references of urease inhibitors for comparison
purposes.
4ꢀNO2ꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀNO2ꢀC6H4
3ꢀNO2ꢀC6H4
3ꢀNO2ꢀC6H4
3ꢀNO2ꢀC6H4
3ꢀNO2ꢀC6H4
3ꢀNO2ꢀC6H4
3ꢀNO2ꢀC6H4
2ꢀNO2ꢀC6H4
2ꢀNO2ꢀC6H4
2ꢀNO2ꢀC6H4
2ꢀNO2ꢀC6H4
2ꢀNO2ꢀC6H4
2ꢀNO2ꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
3ꢀClꢀC6H4
3ꢀClꢀC6H4
3ꢀClꢀC6H4
3ꢀClꢀC6H4
2ꢀClꢀC6H4
143
149
172
192
173
149
104
156
195
173
155
172
156
179
215
216
202
144
120
146
186
135
126
128
182
198
123
126
170
180
103
85
4ꢀMeOꢀC6H4
4ꢀOHꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀClꢀC6H4
C6H5
C6H5CH2
4ꢀMeOꢀC6H4
4ꢀOHꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀClꢀC6H4
C6H5
C6H5CH2
4ꢀMeOꢀC6H4
4ꢀOHꢀC6H4
4ꢀNO2ꢀC6H4
4ꢀClꢀC6H4
C6H5
C6H5CH2
4ꢀMeOꢀC6H4
4ꢀOHꢀC6H4
C6H5
C6H5CH2
4ꢀMeOꢀC6H4
4ꢀOHꢀC6H4
4ꢀOHꢀC6H4
C6H5
C6H5CH2
4ꢀOHꢀC6H4
4ꢀNO2ꢀC6H4
C6H5
C6H5CH2
4ꢀOHꢀC6H4
4ꢀClꢀC6H4
C6H5
Fifty one out of 65 BTUs at 500
extents urease activity in medium reactions containing 10 mM
urea (Table 3). Compounds 10 12 14 16 19 22 37 45
55 57 59 and 60 were the most active BTUs toward jack bean
µM inhibited at different
3
,
4
,
6
,
ꢀ
,
,
,
ꢀ
,
,
,
,
,
urease as attested by percentages of enzyme inhibition equal or
higher than 40% (Table 3). Notably, the effect of BTU 14 on
jack bean urease was comparable to that of the reference
NR
NR
NR
60
inhibitor HU and 3.2ꢀfold higher than that of the inhibitor TU
On the other hand, inhibition lower than 10% categorized the
BTUs 15 30 36 and 41 as poor inhibitors of jack bean urease.
.
61
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
,
,
In fact, in vitro assays with jack bean ureases have been widely
used to assess the potential of several substances as urease
inhibitors of clinical and agricultural interests.10 Eighteen BTUs
were found to be the most active against jack bean type III
urease because they inhibited the ureolytic activity of such
enzyme by 40ꢀ75% (Table 3). Among them, those (12 BTUs)
derived from nonꢀsubstituted benzoyl chloride inhibited the
urease by up to 75% while the ones derived from substituted
benzoyl chloride (except for 37) caused a maximum of 50%
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
3ꢀOMeꢀC6H4
3ꢀOMeꢀC6H4
3ꢀOMeꢀC6H4
3ꢀOMeꢀC6H4
2ꢀOMeꢀC6H4
2ꢀOMeꢀC6H4
177
123
139
96
116
184
145
C6H5CH2
4ꢀMeOꢀC6H4 2ꢀOMeꢀC6H4
4ꢀOHꢀC6H4
4ꢀClꢀC6H4
enzyme inhibition. Indeed, compound 37 that bears
pꢀCl phenyl
2ꢀOMeꢀC6H4
2ꢀOMeꢀC6H4
and ꢀNO2 phenyl groups as cores and , respectively,
m
A
B
inhibited the urease activity by 67% (Fig. 1). As for nonꢀ
substituted benzoyl chloride derived BTUs, the presence of
aValues correspond to the overall yield of twoꢀstep reactions. M.p.,
melting point as compared to that reported elsewhere (if available).
NR, not yet reported so far.
phenyl ring bearing
p
ꢀ
tertꢀbutyl,
o
ꢀNO2 or (
mꢀ or pꢀ)Cl or (mꢀ
or ꢀ)Br groups as the core
p
A seems to contribute to the
effectiveness of BTUs against jack bean urease. These results
corroborate those obtained by Rauf et al. (2013)24 in which the
The BTUs derived from benzoyl chloride and aliphatic amines
or ammonia (
Benzoyl chloride and aromatic amines led to the formation of
BTUs to 26 in yields ranging from 64% to 99% (Table 1).
1 to 4) were obtained in 38ꢀ60% yield (Table 1).
presence of halogen substituents in phenyl ring at core
improved the BTU urease activity. Additionally, previously
reported BTUs bearing ꢀOCOC2H5 phenyl as core and
A
5
p
A
pꢀ
Substitutedꢀbenzoyl chlorides in the presence of a variety of
aromatic amines or benzylamine provided the remainder BTUs
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DOI: 10.1039/C5RA07886E
OCH3 or
m,p-diOCH3 groups as core B were determined as the Michaelian behavior of urease catalysis. Similar behavior was
most active urease inhibitor among the compounds tested.25
observed in assays performed with the other seven BTUs. The
average concentration of urea (KM or KS) that allows for urease
velocity reaching 0.5 Vmax in reactions buffered with phosphate
Table 3. Inhibitory activity of synthesized benzoylthioureas (0.5
mM) toward jack bean urease in reactions containing 10 mM
urea.
(pH 7.4) was 4.2
found to be 10.1
± 0.3 mM and the average Vmax for urease was
+
±
0.3 µ
mol NH4 minꢀ1 mgꢀ1 prot. The
BTU
Urease inhibition (%)
BTU
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
HU
TU
Urease inhibition (%)
LineweaverꢀBurk plots obtained (Fig. 2) indicate that all the
BTUs tested function as mixedꢀtype inhibitors toward urease,
as the lines intersect one with another in the second quadrant.
Mixedꢀtype inhibitors are known to be able to bind to both the
free enzyme (forming EI complex) and enzymeꢀsubstrate
complex (forming ESI complex), therefore being associated to
two equilibrium dissociation constants (Ki for the former and
Ki’ for the latter complexes). In fact, the equilibrium
dissociation constants related to complexes former among
urease, urea and each BTU individually (Ki values) were found
to be higher than the equilibrium dissociation constants for
complexes formed between urease, urea and the corresponding
BTU (Ki’ values) (Table 4). These results point out that the
affinity of the BTUs object of this study for the urease active
site is higher than that of allosteric site(s). The BTU 12 was the
least potent with respect to the binding to either the active site
or allosteric site(s) of urease. Some unsymmetricallyꢀ
substituted N,N’ꢀdiaryl thiourea, compounds structurally related
to BTUs, were recently described as mixed inhibitors of jack
bean urease.38
1
2
3
4
5
6
7
8
32.0
11.2
43.8
44.0
24.2
47.4
27.0
17.7
31.7
40.7
51.7
64.2
27.3
73.9
6.4
49.1
24.1
32.2
60.0
65.2
51.0
57.2
33.4
19.7
NA
NA
8.7
66.9
NA
29.2
NA
3.8
NA
NA
17.7
47.6
NA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
NA
13.8
37.5
NA
28.4
26.5
33.0
20.1
39.1
17.6
43.5
30.6
41.6
49.3
22.3
12.6
15.2
29.6
10.8
73.6
22.6
NA
17.9
NA
NA
5.1
28.3
NA
16.3
NA
Table 4. Effect of benzoylthioureas (BTUs) on jack bean urease
kinetics. Urea (1 to 32 mM) was incubated for 5 min with jack
bean Type III urease in the absence or presence of BTU (250 to
500
µM). The KM (or KS) and Vmax values determined were,
+
respectively, 4.2
±
0.3 mM and 10.1
±
0.3
µ
mol NH4 minꢀ1 mgꢀ1
protein.
NA, nonꢀactive with respect to the ability to inhibit urease activity
when tested at our experimental conditions. Hydroxyurea (HU) and
thiourea (TU) were used as references of urease inhibitor.
BTU
11
12
14
19
20
21
22
37
aKi (µM)
283.7 ± 25.8
1,776.6 ± 670.0
166.9 ± 66.0
396.7 ± 11.1
475.2 ± 166.0
795.6 ± 90.4
308.7 ± 91.8
416.9 ± 100.1
aKi’ (µM)
631.4 ± 65.1
5,170.8 ± 524.0
380.2 ± 83.2
1,325.4 ± 130.2
824.4 ± 169.9
1,211.9 ± 117.1
1,257.5 ± 99.8
1,349.9 ± 379.6
Effect of BTUs on kinetic parameters of jack bean type III
urease
Assays using fixed concentrations of BTUs in reactions
containing urea in the range of 1 ꢀ 32 mM were performed to
expand the knowledge on the type of enzyme inhibition
triggered by the most active BTUs synthesized. The BTUs that
aKi corresponds to the equilibrium dissociation constant for ureaseꢀ
BTU complex while Ki’ is the equilibrium dissociation constant for
ureaseꢀureaꢀBTU complex. Values are the mean ± standard
deviation.
caused urease inhibition by over 50%, namely 11, 12, 14, 19ꢀ22
and 37 (Table 3), were then selected for the studies of
mechanism of urease inhibition. Reactions free of inhibitor
were carried out whenever a BTU would be tested since slight
changes in reaction conditions may affect the kinetic
parameters. A Vo versus urea concentration plot obtained from
data of assays with 11 is shown in Figure 2 to exemplify the
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1.2
400
300
µM
BTU 11
9.0
7.5
6.0
4.5
3.0
1.5
0.0
I
-free
µM
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
300
400
µM
V
µ
M
I-free
o
V
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
5
10
15
20
25
30
35
Urea (mM)
1/[Urea]
2.4
2.0
1.6
1.2
0.8
0.4
0.0
1.0
BTU 14
BTU 19
400
300
µ
M
0.8
0.6
0.4
0.2
0.0
450
µ
M
350µ
M
µM
V
V
I-free
I-free
0.0
0.2
0.4
0.6
0.8
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1/[Urea]
1/[Urea]
1.4
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
BTU 37
BTU 22
450
350
µM
1.2
1.0
0.8
0.6
0.4
0.2
0.0
350
250
µM
µM
µM
V
V
I-free
I-free
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1/[Urea]
-0.2
1/[Urea]
Fig. 2. Representative MichaelisꢀMenten hyperbola and LineweaverꢀBurk plots for jack bean urease in the presence of the
benzoylthioureas (BTUs). Increasing concentrations of urea were incubated for 5 min with jack bean Type III urease in the absence of
inhibitor (Iꢀfree) or presence of BTU at indicated concentrations. A Vo versus urea concentration plot obtained from data of assays
with 11 is shown to exemplify the Michaelian behavior of urea catalysis.
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ARTICLE
60
Inhibition of soil ureases by BTUs
b
50
40
30
20
10
0
The results obtained from in vitro assays encouraged us to
further investigate the potential of BTUs as urease inhibitors of
agricultural interest against a soil used for crop production in
Brazilian Cerrado. For these experiments,
thiophosphoric triamide (NBPT), an additive in ureaꢀbased
b
b
b
b
b
a
a
Nꢀ(butyl)
a
a
a
fertilizers, was used as a reference of urease inhibitor.
a
a
a
a
Fig. 3 presents the results for 28 BTUs, found to inhibit the
activity of soil ureases by at least 15%. Analysis of variance
(oneꢀway ANOVA) followed by Stepwise contrast test allowed
to separate the BTUs in two groups of inhibitors in which the
first one included the compounds more potent than NBPT
Compounds tested
(BTUs 3, 6, 10, 12, 16, 19 and 22; P < 0.001) and the second
one comprised 21 compounds found to be as potent as NBPT
(Fig. 3). Other 19 BTUs inhibited soil ureases in the range from
60
50
40
30
20
10
0
5% to 10% while compounds
50 58 65 failed to inhibit the formation of NH4 catalyzed by
soil ureases.
7, 8, 13, 23, 28, 36, 40, 46, 47,
+
,
ꢀ
b
a
a
Although jack bean urease shares high homology of amino acid
sequence with ureases of other organisms12,40, contrasting
results were obtained when comparing in vitro findings with
a
a
a
a
a
a
a
a
a
a
a
a
those obtained from soil. For instance, while BTUs 26, 32, 34
and 38 where inactive in in vitro assays (Table 3), they were as
effective as NBPT to inhibit soil ureases (Fig. 3). Then, it
becomes evident that results from in vitro assays per se are not
enough to select potential urease inhibitors of agricultural
interest. However, assays performed with purified urease are
important tools to pursue a better comprehension on the
mechanism of action of a certain inhibitor as well as the
structural features critical for the design of more potent
inhibitors. Moreover, the complex physicochemical and
biological nature of soil matrix may culminate in the
metabolization of xenobiotic substances as is the case of
synthetic urease inhibitors. Chemical transformations triggered
by soil microbiota may result in lossꢀ or gainꢀofꢀfunction of a
certain xenobiotic.
1
2
6
1
2
4
7
8
9
9
2
5
6
7
T
2
2
2
3
3
3
3
3
3
4
5
5
5
5
P
B
N
Compounds tested
Fig. 3. Benzoylthioureaꢀtriggered inhibition of soil ureases.
Amended soil (0.5 g) was incubated with urea (72 mM) in the
presence or absence of compoundsꢀtest at 0.5 mM. Results are
representative of independent experiments, each done in
quadruplicate or quintuplicate. Standard errors were lower than
11%. NBPT, reference soil urease inhibitor. Distinct letters
indicate significant difference (P < 0.001) according to variance
analysis (oneꢀway ANOVA) followed by Stepwise contrast test.
Thermal stability of BTUs
The BTUs that clustered into the group of the best soil urease
inhibitors (3, 6, 10, 12, 16, 19 and 22) were then selected for
assessing their thermal stability. The thermogravimetric curves
clearly show that all BTUs tested are more thermal stable than
NBPT (Fig. 4). The event of mass loss for compounds 10 (most
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stable BTU) and 22 (less stable BTU) begins at 210 oC and 166 Novel Urease Inhibitors (www.redniu.org) which is financially
oC, respectively, while NBPT starts to lose mass at 151 oC (Fig. supported by the Brazilian agencies CNPq, CAPES and
4). The other BTUs (3, 6, 12, 16 and 19) show a weight loss at FAPEMIG. LVM and AF are recipients of research fellowships
o
o
temperature range from 180 C to 195 C. Other BTUs are from CNPq.
reported to be resistant to heat.25,41,42
Notes and references
100
80
60
40
20
0
aDepartamento de Química, Centro de Ciências Exatas, Universidade
Estadual de Londrina, Londrina, PR, Brazil
bDepartamento de Botânica, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
cDepartamento de Fitotecnia, Centro de Ciências Agrárias, Universidade
Federal de Roraima, Boa Vista, RR, Brazil.
dDepartamento de Química, Instituto de Ciências Exatas, Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
†These authors contributed equally to this work.
NBPT
10
*Corresponding authors. F. Macedo Jr. (Email: macedofc@uel.br; Tel:
+55ꢀ43ꢀ3371ꢀ4811) and L.V. Modolo (Eꢀmail: lvmodolo@icb.ufmg.br;
Tel: +55ꢀ31ꢀ3409ꢀ3008).
22
3,6,12,16,19
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See DOI:
10.1039/b000000x/
50 100 150 200 250 300 350 400 450 500 550 600
Temperature (°C)
Fig. 4. Thermogravimetric behavior of the most promising soil
urease benzoylthiourea inhibitors. NBPT was used as reference
1.
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.
2
3
Conclusions
4
5
The design, synthesis and biological evaluation of 65
benzoylthioureas (BTUs) allow for the in vitro identification of
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urease inhibition. Screening on tropical soil revealed 28 BTUs
as promising soil urease inhibitors, being compounds
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3, 6, 10,
,
,
6
7
8
i
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Maize and Sorghum, Brazil) for providing with soil samples
and corresponding physicochemical characterization data, Dr.
Maria I. Yoshida (UFMG, Brazil) for assistance with
thermogravimetric analysis and MSc. Matheus L. Souza
(UFMG, Brazil) for helping with statistical analysis. This work
was made possible by the Network for the Development of
12
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DOI: 10.1039/C5RA07886E
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This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 9

RSC Advances
Page 10 of 10
Mechanism of urease inhibition
1.4
BTU 37
450 M
X
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
H2N
NH2
350 M
I-free
X = O Urea (urease substrate)
X = S Thiourea (urease inhibitor)
S
O
A
N
N
B
H
H
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1/[Urea]
Benzoylthioureas (BTUs)
(Potential new inhibitors)
65 BTUs
synthesized
Inhibitors of agricultural interest
60
50
40
30
20
10
0
A
H
CH3
CH2CH3
b
tert-Butyl
b
b
b
b
b
a
a
R = H, CH3, tert-butyl, MeO,
NO2, OH, Br or Cl
a
a
a
N
a
a
a
a
N
N
R = H, MeO, NO2 or Cl
B
1
2
3
4
5
6
T
10 12 15 16 17 18 19 20
P
B
N
Compounds tested